We study the molecular mechanisms involved in assembly and function of translation initiation complexes involved in protein synthesis, using budding yeast as a model system to exploit its powerful combination of genetics and biochemistry for dissecting complex cellular processes in vivo. The translation initiation pathway produces an 80S ribosome bound to mRNA with methionyl initiator tRNA (tRNAi) base-paired to the AUG start codon. The tRNAi is recruited to the 40S subunit in a ternary complex (TC) with GTP-bound eIF2 to produce the 43S preinitiation complex (PIC) in a reaction stimulated by eIFs 1, 1A, 3 and 5. The 43S PIC attaches to the 5' end of mRNA, facilitated by cap-binding complex eIF4F (comprised of eIF4E, eIF4G, and RNA helicase eIF4A) and PABP bound to the poly(A) tail, and scans the 5 untranslated region (UTR) for the AUG start codon. Scanning is promoted by eIFs 1 and 1A, which induce an open conformation of the 40S, and by eIF4F and other RNA helicases, such as Ded1, that remove secondary structure in the 5' UTR. AUG recognition by tRNAi evokes irreversible hydrolysis of the GTP bound to eIF2, dependent on the GTPase activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC and tRNAi into the ribosomal P site. After joining of the 60S subunit, producing the 80S initiation complex, the eIF2-GDP is recycled to eIF2-GTP by guanine nucleotide exchange factor (GEF) eIF2B for the next round of translation initiation. Enhanced eIF1 binding to the 40S ribosome impedes conformational rearrangements of the preinitiation complex and elevates initiation accuracy. Recognition of the start codon is thought to require dissociation of eIF1 from the 40S subunit, enabling Pi release by the TC, rearrangement of the 40S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. Supporting this model, we showed previously that eIF1 mutations that impair its binding to 40S subunits in vitro increase initiation at UUG codons (Sui- phenotype) and derepress translation of GCN4 mRNA (Gcd- phenotype), indicating impaired TC loading. This indicated that direct contact of eIF1 with 18S rRNA is crucial for eIF1s ability to stabilize the open conformation of the 40S subunit competent for rapid loading of TC in a conformation capable of sampling triplets entering the P site (POUT), while blocking accommodation of tRNAi in the PIN state required for AUG recognition. Recently, we selected eIF1 Ssu- mutations that suppress the elevated UUG initiation and reduced rate of TC loading in vivo conferred by aforementioned eIF1 Sui- substitutions. Importantly, several such Ssu- substitutions increase eIF1 affinity for 40S subunits in vitro, and the strongest-binding variant (D61G), predicted to eliminate ionic repulsion with 18S rRNA, both reduces the rate of eIF1 dissociation and destabilizes the PIN state of TC binding in PICs reconstituted with Sui- variants of eIF5 or eIF2. Thus, eIF1 dissociation from the 40S subunit is required for the PIN mode of TC binding and AUG recognition, and increasing eIF1 affinity for the 40S subunit increases initiation accuracy in vivo. Conserved residues in yeast initiator tRNA calibrate initiation accuracy by regulating preinitiation complex stability at the start codon. Eukaryotic tRNAi contains unique sequence features, but their importance in start codon selection was unknown. We found that disrupting the conserved C3:G70 base pair in the acceptor stem enhances initiation at UUG codons (Sui- phenotype), and also reduces the rate of TC binding to 40S subunits both in vitro and in vivo (Gcd- phenotype). These defects are suppressed by an Ssu- substitution in the eIF1A SI element shown previously to stabilize the open/POUT conformation. Because the consequences of C3:G70 substitutions mimic the known effects of Sui- mutations in eIF1 and eIF1A SE elements, it appears that the C3:G70 base pair functionally interacts with eIF1 and eIF1A to promote the POUT conformation, favoring rapid TC binding and impeding rearrangement to the PIN conformation at non-AUG codons. Substituting the conserved G31:C39 base pair in the anticodon stem with different base pairs also reduces initiation accuracy (Sui- phenotype) but does not impair TC binding to the PIC, suggesting that base pair replacements at 31:39 enhance UUG initiation by stabilizing the PIN state at near-cognate triplets rather than destabilizing the POUT conformation. By contrast, mutations that eliminate base pairing at 31:39 have the opposite effect of increasing initiation accuracy (Ssu- phenotype), which is suppressed by a Sui- substitution of T-loop residue A54 in tRNAi. These opposing genetic phenotypes are paralleled by opposite effects of the Sui- and Ssu- substitutions on the stability of Met-tRNAi binding to reconstituted PICs in vitro. We propose that any of the 3 alternative Watson-Crick base pairs at position 31:39 is sufficient to stabilize PIN and promote initiation by inherently less stable PICs formed at UUG codons. However, the (wild-type) G31:C39 pair uniquely imposes an energetic penalty on the PIN state that is compensated effectively only with the perfect codon:anticodon duplex at AUG codons. Thus, conserved bases throughout tRNAi, from the anticodon stem to acceptor stem, play key roles in ensuring the efficiency and fidelity of start codon recognition in vivo. Identification and characterization of functionally critical, conserved motifs in the internal repeats and N-terminal domain of yeast translation initiation factor 4B (yeIF4B). eIF4B stimulates recruitment of mRNA to the 43S PIC, but its molecular function is unclear. The yeast (y)eIF4B contains an unstructured N-terminal domain (NTD), RNA-recognition motif (RRM), and a domain comprised of seven imperfect repeats of 26 amino acids; and previous studies had implicated the RRM and its RNA binding activity in promoting translation initiation. By analyzing the effects of deletions and mutations of yeIF4B domains on PIC attachment to mRNA in vitro and translation initiation in vivo, we found that the 7-repeats domain is critical for productive interaction with the PIC and other components of the initiation machinery, particularly eIF4A, in promoting PIC attachment to mRNA. The NTD also plays a role in accelerating mRNA binding to the PIC but, surprisingly, the RRM and its associated ssRNA binding activity are dispensable in vitro and in vivo. We recently determined that only 2 of the 7 internal repeats are sufficient for wild-type (WT) yeIF4B function in vivo when all other domains are intact, whereas at least three are needed in the absence of the NTD or when functions of eIF4F components are compromised. We corroborated these observations demonstrating that yeIF4B variants with only one or two repeats display substantial activity in promoting mRNA recruitment by the PIC in vitro, whereas additional repeats are required at lower levels of eIF4A or when the NTD is missing. We also demonstrated that only three highly conserved positions in the 26-aa repeat are essential for function in vitro and in vivo, and identified conserved motifs in the NTD and demonstrated functional overlap of two such motifs. These results provide a comprehensive description of the critical sequence elements in yeIF4B that support eIF4F function in mRNA recruitment by the PIC.